The Rac1 GTPase plays a pivotal role in the pathogenesis of ischemic and reperfusion injury. Rac1 is activated during ischemia/hypoxia and reoxygenation/reperfusion, and by leading to the production of reactive oxygen species (ROS) promotes injury and inflammation. However, the molecular mechanisms leading to the activation of racl during hypoxia/ischemia and reoxgenation/reperfusion are not known. P66shc belongs to the shcA family of adaptor proteins. It is unique among she proteins by virtue of its ability to sense extracellular oxidative stimuli and accordingly govern intracellular levels of reactive oxygen species(ROS). The mechanism by which p66shc regulates intracellular ROS levels is not fully characterized, and the importance of p66shc in mediating oxidative stress during hypoxia/ischemiaand reoxygenation/reperfusion is not known. Based on novel preliminary data, this Project advances the concept that there exists a fundamental relationship between Rac1 and p66shc, and that this relationship is a primary determinant of racl activity and ROS production during ischemia/hypoxia and reperfusion/reoxygenation. We hypothesize that ischemia/hypoxia leads to an increase in expression and activity of p66shc, which, in turn, promotes ROS generation by stimulating the activity of the racl GTPase. Proposed experiments will examine the role of p66shc in ROS production and cardiovascular injury, the molecular mechanism through which p66shc activates racl, and the molecular regulation of p66shc expression, during cardiovascular ischemia/hypoxia and reperfusion/hypoxia. These studies will be performed at the molecular, cellular, and whole animal levels, using gene transfer, and gene knockdown methods, and should therefore provide an in-depth understanding about the importance of p66shc, and its mechanisms, in ischemic and post-ischemic cardiovascular injury.